Set of means for treating a malignant pathology, an autoimmune disease or an infectious disease

ABSTRACT

Kit of parts for treating a malignant pathology, an auto-immune disease or an infectious disease, comprising an effector cell which expresses the FcγRIII receptor (CD16) on its surface, and a monoclonal antibody, the affinity of the Fc region of said monoclonal antibody for CD16 being greater than the affinity of the Fc region of the polyclonal immunoglobulins for CD16.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Entry of International ApplicationNo. PCT/FR2008/000598, filed on Apr. 25, 2008, which claims priority toFrench Patent Application 07 03 013, filed on Apr. 25, 2007, both ofwhich are incorporated by reference herein.

BACKGROUND

The present invention relates in general to treating a malignantpathology, an auto-immune disease, or an infectious disease, especiallyby means of an effector cell which expresses an FcγR receptor on itssurface.

Used more and more in research, antibodies are also tools of choice indiagnostics and therapeutics, where they represent an alternative toconventional treatments. Numerous preparations of antibodies fortherapeutic usage, of plasmatic origin or originating frombiotechnologies, are currently on the market, or in clinical developmentphase. Their properties are exploited to produce therapeutic toolscapable of binding specifically with their target, and efficientlyrecruiting the cells of the immune system.

Research has recently focussed on improving the efficiency ofantibodies, and more particularly on manipulation of their constant Fcregion. It is the latter which is responsible for the “effector”properties of antibodies, since it allows mobilisation of the effectorcells of the immune system and of proteins of the complement. Thisfaculty is made possible by the presence, on certain effector cells, ofglycoproteins i.e. the receptors Fc (FcR). These receptors are capableof binding the constant region of the antibodies, once the latter arefixed, by their variable region, on the target antigen. The binding ofthe Fc region of antibodies on the FcR carried by the effector cellscauses in the latter activation of cytotoxic mechanisms such asphagocytosis and ADCC (cellular cytotoxicity dependent on antibodies orAntibody-Dependent Cell-mediated Cytotoxicity). During an auto-immunedisease, the immune system, the natural role of which is to protect theorganism from aggression, causes an inflammatory response in the absenceof extraneous bodies and thus itself causes tissue lesions by“accidentally” attacking the molecules of the self. There are differentauto-immune pathologies affecting different tissues or differentfunctions of the body. For example, the brain is affected in thosepeople suffering from multiple sclerosis, the intestines are the targetin patients afflicted with Crohn's disease and synovia, bones andcartilages are affected in those afflicted by rheumatoid polyarthritis.

During development of the auto-immune disease, several phenomena mayeventuate, such as progressive destruction of one or more types oftissue, abnormal growth of an organ or modifications of the function(s)of the organ affected. The tissues or organs most often affected duringan auto-immune disease are haematopoietic cells, blood vessels,connective tissues, endocrine glands, muscles, joints and skin.Auto-immune diseases are often associated with a chronic inflammatorypathology. The most frequent case is represented by rheumatoidpolyarthritis and juvenile rheumatoid arthritis which are two types ofinflammatory arthritis. Arthritis is a general term designatinginflammation of joints.

The plurality of treatments has numerous secondary effects or do notfully prevent progression of the disease. There is currently no idealtreatment and none to help cure patients, culminating in an obvious needfor novel therapies which are more efficient and above all curative.Since B lymphocytes (LB) are those cells producing auto-antibodies oftenresponsible for the development of auto-immune diseases, theirdestruction by administration of a specific monoclonal antibody of thiscellular type may be only beneficial to patients, as shown for rituximabrecently approved for treating rheumatoid polyarthritis.

Also, despite considerable progress made in improving sanitaryconditions, in immunisation and in anti-microbial therapies, infectiousdiseases represent a persistent and significant problem for modernmedicine. The most widespread disease, the simple cold, is an infectiousdisease in the same category as AIDS (acquired immune deficiencysyndrome), the most feared disease. It has been proven that certainneurological disorder classes such as degenerative diseases were in factassociated with infection.

Infectious diseases are set to remain a major medical problem in thefuture. During an infectious disease, the monoclonal antibodies may playtwo complementary roles: a neutralising role of the pathogenic agent ortoxins secreted during the acute phase of infection and a destroyingrole of reservoir cells during transition to the chronic phase.

The destruction of host cells enabling low-noise duplication of thepathogenic agent could prevent passage to a chronic phase ending mostoften in the development of serious pathologies such as an auto-immunedisease or a cancer. These days, despite the existence of a real need,there is almost no efficacious anti-infectious treatment in thetreatment of chronic phases. On the other hand, the beneficial effectsof small molecules (antibiotic, anti-parasitic, anti-viral) during theacute phases of infections are becoming increasingly compromised by thedevelopment of cross-resistance. Therefore the appearance of bacteriamulti-resisting to antibiotics poses a problem of public health with 6%to 7% of all hospitalisations complicated by more or less seriousnosocomial infection, or around 750,000 cases of the 15 millionshospitalisations annually(http://www.senat.fr/rap/r05-21/r05-4211.html#toc13). In total,nosocomial infections would thus be responsible of 9000 deaths everyyear, whereof 4200 concern patients for whom the vital prognosis was notengaged in the short term on their entering hospital(http://www.senat.fr/rap/r05-421/r05-4213.html). Therefore, it appearsnecessary to develop innovative drugs which will offer a therapeuticalternative for doctors and their patients.

A tumour corresponds to a neoplasic mass resulting from uncontrolledproliferation of cells which may be benign or malignant. Benign tumoursgenerally remain localised. Malignant tumours are collectively calledcancers. The term “malignant” in general means that the tumour iscapable of invading and destroying adjacent structures and diffusingtowards remote sites, in the long run causing the death of the patient(Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co.,Philadelphia, pp. 68-122). Cancer may arise in numerous differentlocations and behave differently as a function of its tissular origin.Currently, means for treating cancers are surgery, chemotherapy,hormonal therapy and/or irradiation for eradicating tumour cells presentin the patient (Stockdale, 1998, “Principles of Cancer PatientManagement”, in Scientific American Medicine, vol. 3, Rubenstein andFederman, Eds., Chapter 12). All these treatments have major drawbacks.For example, despite the availability of a large variety of chemicalmolecules, chemotherapy causes numerous side-effects, such as severenausea, medullar aplasia, immunosuppression, etc. The gravity of some ofthese effects obliges the doctor to sometimes discontinue treatment. Inaddition, despite administration of a combination of several chemicalmolecules, most of tumour cells is resisting or develop resistance tochemotherapy agents. In fact, cells resisting to a particular agentadministered in the current protocol, are unfortunately also resistingto other drugs, including those acting via a mechanism different to thatused by the agent administered in the treatment protocol. Thisphenomenon, known under the name of multidrug resistance, is often theorigin of the therapeutic failure of standard chemotherapy protocols.There is thus a real need for innovative anticancer therapies, inparticular for treating cancers refractory to conventional treatmentsuch as surgery, irradiation, chemotherapy or hormonal therapy.

A promising alternative is immunotherapy, in which the tumour cells arespecifically targeted by the antibodies which are specific for tumorousantigens. Major efforts have been made to exploit the specificity of theimmune response, for example hybridome technology has enabled thedevelopment of monoclonal antibodies which are specific for antigensexpressed by tumour cells (Green M. C. et al., 2000 Cancer Treat Rev.,26: 269-286; Weiner L. M., 1999 Semin Oncol. 26 (suppl. 14):43-51). Thedestruction of harmful cells of the host or pathogenic agentscorresponds to the desired efficiency mechanism of monoclonal antibodiesirrespective of the targeted pathology. It is thus critical for theseantibodies to be improved so as to interact optimally with the effectorcells of the immune system of the patient.

Chronic lymphoid leukaemia B (LLC-B), a disease characterised bymalignant proliferation of B lymphocytes (LB), is the most frequent formof leukaemia. Current treatment is essentially based on therapeuticabstinence for the early stages of the disease. In the event of clinicalor haematological symptomatology, patients are classically treated bycorticoids alone or in association with anti-mitotic molecules. For mostof patients, resistance to treatment sets in more or less long term andgenerally ends in the failure of the therapeutic effort with theappearance of chemo-resisting cells. Chemotherapy is responsible forsubstantial side effects, especially with myelotoxicity generating animmune deficit responsible for the appearance of serious infections,sometimes deadly, in patients. Many therapeutic approaches focussed ondestroying tumorous B cells as specifically as possible have beenevaluated. The specific expression of the CD20 molecule by the tumorous(and normal) LB has allowed development of therapies based on the use ofhuman anti-CD20 monoclonal antibodies.

A single non-radio labelled anti-CD20 monoclonal antibody, rituximab(Rituxan®, Genentech and Mabthéra™, Roche), is currently availablecommercially. It is indicated for treating patients affected byfollicular lymphomas of stage III-IV and in association withchemotherapy for treating patients presenting diffuse CD20 positivelarge B cells aggressive non-Hodgkin's lymphoma (NHL). Since itsefficiency remains variable and often modest when used as single agent(Teeling et al. 2004, Blood 104 (6)1793-800), it is the most often usedin association with chemotherapy.

Rituximab has also been evaluated in patients with LLC-B. This antibodyhaving presented only slight efficiency when used in monotherapy, it iscurrently administered in association with chemotherapy. Many reasonsmay explain the failure of monotherapy by rituximab in patients affectedby LLC-B: first, rituximab in vitro causes slight ADCC activity on Bcells, and, contrary to normal LB and in NHL, LB of LLC-B express onlyfew CD20 molecules on their surface (density about 5 times less,quantitative measure by flow cytometry), thus limiting the quantity ofantibodies on the cellular surface and thus the associated cytotoxicfunctions (ADCC and activity complement especially). It is thus of majorimportance to focus on alternative therapies including antibodies whichare specific for the CD20 antigen and capable of efficiently causinglysis in tumour cells, including those slightly expressing the antigen.

Macrophages, effector cells of inherent immunity, play a major role inanti-tumorous responses. Naturally present in an inactive form (in theabsence of any pathology), they may be activacted in vivo or in vitro bydifferent routes, such as ingestion of pathogens or binding to receptorsexpressed at the surface of immune complexes (binding to FcR via the Fcregion of antibodies) or cytokines, immuno-modulatory molecules producedespecially during an inflammatory phenomenon. Activation induces lyticand thus increased anti-tumorous activity in macrophages (Adams D. andHamilton T.: Activation of macrophages for tumour cell kill: effectormechanism and regulation. In Heppner & Fulton (Eds), Macrophages andcancer. CRC Press, 1988, p. 27; Fidler I.: Macrophages and metastases. Abiological approach to cancer therapy. Cancer Res, 45: 4714, 1985).

In addition, macrophages, or other cells derived from monocytes or theirprecursors, are antigens presenting cells. Due to their strong capacityfor endocytosis, digestion and presentation to T lymphocytes ofantigenic peptides associated with molecules of the of majorhistocompatibility complex (MHC), they are capable of inducing aspecific immune response.

With the aim of increasing the efficiency of rituximab, its associationwith macrophages activated ex vivo in the presence of interferon-γ(IFN-γ) was evaluated in vitro in a test for lysis of primary cells ofLLC-B (Lefebvre M L, Stefan W. Krause, Salcedo M, Nardin A. Ex vivoactivated human macrophages kill chronic lymphocytic leukemia cells inthe presence of Rituximab: mechanism of antibody-dependent cellularcytotoxicity and impact of human serum. J. Immunother; vol. 29, no. 4:388-397, 2006). The results indicate that the strong lysis of LLC-Bcells by activated macrophages in the presence of rituximab is stronglyinhibited by increasing concentrations of human serum. This inhibitionis linked to competition by polyclonal immunoglobulins which are presentin the serum vis-à-vis the binding of the rituximab-LLC-cell complex tothe various FcR expressed at the surface of the macrophages. Theintensity of this inhibition depends on the used concentrations ofrituximab and effector cells: target cells ratio (Effector:Target orE:T). Despite the existence of numerous therapeutic tools for treatingcancers, auto-immune diseases, or infectious diseases, there is still asubstantial need for novel immunotherapy products, evidencing greaterefficiency and greater safety than existing products.

SUMMARY

A primary object of the invention is a kit of parts for treating amalignant pathology, an auto-immune disease or an infectious disease,comprising an effector cell which expresses the FcγRIII receptor (CD16)on its surface, and a monoclonal antibody, in which the affinity of theFc region of said monoclonal antibody for CD16 is greater than theaffinity of the Fc region of the polyclonal immunoglobulins for CD16.Advantageously, the effector cell which expresses the FcγRIII receptor(CD16) on its surface is a monocyte or a cell derived from a monocyte ora monocyte precursor which expresses the FcγRIII receptor (CD16) on itssurface. Advantageously, this cell is selected from monocytes expressingCD16, macrophages, Natural Killer cells (NK), dendritic cells andperipheral blood mononuclear cells as a whole (Peripheral BloodMononuclear Cell or PBMC). Advantageously, the cell expressing CD16 onits surface is selected from monocytes expressing CD16, macrophages anddendritic cells.

More particularly, the monocyte derived cell a monocyte precursor, whichexpresses CD16 on its surface, is a macrophage. Advantageously, themonoclonal antibody is not displaced by polyclonal immunoglobulins,particularly those present in the serum, due to the high affinity of theFc region of said monoclonal antibody for CD16. Advantageously, themonoclonal antibody binds to CD16 of said monocyte or monocyte precursorderived cell with an affinity greater than 2.10⁶M⁻¹.

In a particularly advantageous manner, the monoclonal antibody isproduced in the form of a monoclonal antibodies composition, in whicheach antibody has sugar chains bound to N at the Fcγ glycosylation site(asparagine 297, according to Kabat), and in which, among all sugarchains which are bound to N at said glycosylation site of all theantibodies of said composition, the fucose rate is less than 65%. In aparticular embodiment of the invention, the monoclonal antibody isdirected against an antigen selected from antigen 5C5 (tumorous antigenexpressed by the cells of renal carcinomas), BCR (B Cell Receptor), anidiotype such as that of anti-FVIII inhibitor antibodies, TCR (T CellReceptor), CD2, CD3, CD4, CD8, CD14, CD15 CD19, CD20, CD21, CD22, CD23,CD25, CD45, CD30, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66(a, b, c, d), CD74, CD80, CD86, CD126, CD138, CD154, MUC1 (Mucine 1),MUC2 (Mucine 2), MUC3 (Mucine 3), MUC4 (Mucine 4), MUC16 (Mucine 16),HM1.24 (specific antigen for plasmocytes which is overexpressed inmultiple myelomas), tenascin (protein of the extra-cellular matrix), GGT(gamma-glutamyltranspeptidase), VEGF (Vascular Endothelial GrowthFactor), EGFR (Endothelial Growth Factor receptor), CEA(carcinoembryonic antigen), CSAp (colon-specific antigen-p), ILGF(Insulin-Like Growth factor), placental growth factor, Her2/neu,carbonic anhydrase IX, IL-6, proteins S100 (multigenic family ofproteins binding to calcium), MART-1 (tumorous differentiation antigenassociated with melanoma), TRP-1 (tyrosinase-related protein 1), TRP-2(tyrosinase-related protein 2), gp100 (glycoprotein 100 kDa), amyloidproteins, rhesus D antigen, MHC molecules of class I and II such asHLA-DR), an antigen resulting from the expression of mutated genes,especially oncogenes or tumour-suppressor genes, an antigen derived fromoncogene virus which are expressed by certain well defined tumours, aubiquitous antigen overexpressed in some tumours and slightly expressedin some normal tissues, such as for example the type II receptor of theMüllerian hormone, a glycosylated or non-glycosylated protein, aphospholipid, a molecule of the self or of the non self expressed orexposed on the membrane by infected cells such as phosphatidylserine,and a protein expressed or secreted by a pathogenic agent (bacterialtoxin, protein complexes of the bacterial or parasitic wall, viralenvelope glycoproteins, for example from HIV virus, HBV, HCV and RSV,etc.).

Preferably, the monoclonal antibody is directed against CD20. In apreferred embodiment of the invention, the anti-CD20 antibody isproduced by the cell line R509 deposited to the CNCM on Nov. 8, 2004under the accession number I-3314, or by the cell line R603, depositedto the CNCM on Nov. 29, 2005 under accession number I-3529 (CNCM:Collection Nationale de Culture de Microorganismes, Institut Pasteur, 25rue du Docteur Roux, 75724 Paris Cedex 15-France). Advantageously, thekit of parts of the invention is intended for use in therapy,simultaneously, sequentially or separately. Advantageously, in the kitof parts of the invention, the effector cell expressing CD16 on itssurface has a cytotoxic activity on the cell targeted by said antibody,which is favoured by the interaction of the antibody with CD16.Advantageously, in the kit of parts of the invention, the monoclonalantibody induces cytotoxicity by ADCC activity or by phagocytosis ofsaid antibody targeted cell in the presence of an effector cellexpressing CD16.

Another object of the invention is a pharmaceutical compositioncontaining the kit of parts according to the invention, andpharmaceutically acceptable excipients. Another object of the inventionrelates to the use of the kit of parts of the invention formanufacturing a drug.

Another object of the invention relates to the use of the kit of partsof the invention for manufacturing a drug intended for treatment of amalignant pathology.

Advantageously, the malignant pathology is selected from solid tumoursand malignant haemopathies. Advantageously, the solid tumours areselected from melanomas, carcinomas, sarcomas, gliomas and skin cancers.Advantageously, the carcinomas are selected in the group consisting ofkidney, breast, oral cavity, lungs, gastro-intestinal tract, ovaries,prostate, uterus, bladder, pancreas, liver, gallbladder, skin andtesticles carninomas. Advantageously, malignant haemopathies areselected from lymphoproliferative, myeloproliferative, myelodysplasicsyndromes and acute myeloid leukemias with, for example, type B NHL,acute or chronic B lymphoid leukemias, Burkitt's lymphoma,tricholeucocyte leukaemia, acute and chronic myeloid leukemias, Tlymphomas and leukemias, Hodgkin's lymphomas, Waldenström'smacroglobulinemia and multiple myelomas.

Another object of the invention relates to the use of the kit of partsof the invention for manufacturing a drug for the treatment of anauto-immune and/or of a primitive or secondary inflammatory disease,which is specific to organs or systemic and which is associated or notwith pathogenic auto-antibodies. Advantageously, the auto-immune and/orinflammatory disease is selected from organ graft rejection, or graftversus host disease, rheumatoid polyarthritis, disseminated lupuserythematosus, sclerodermia, primitive Sjögren's syndrome (orGougerot-Sjögren syndrome), auto-immune polyneuropathies such asmultiple sclerosis, type I diabetes, auto-immune hepatitis, ankylosingspondylarthritis, Reiter's syndrome, gout arthritis, coeliac disease,Crohn's disease, Hashimoto's thyroiditis, Addison's disease, auto-immunehepatitis, Basedow's disease, ulcerative colitis, vasculitis such assystemic vasculitis associated with ANCA (antineutrophil cytoplasmicantibody), auto-immune cytopenias and other haematological complicationsin adults and children, such as acute or chronic auto-immunethrombopenias, auto-immune haemolytic anaemias, haemolytic disease ofthe newborn (HDN), cold agglutinin disease, thrombocytopenic thromboticpurpura and acquired auto-immune haemophilia; Goodpasture's syndrome,extra-membraneous nephropathies, auto-immune bullous skin disorders,refractory myasthenia, mixed cryoglobulinemias, psoriasis, juvenilechronic arthritis, inflammatory myositis, dermatomyositis and childrensystemic autoimmune diseases including the antiphospholipide syndrome.

Another object of the invention relates to the use of the kit of partsof the invention for manufacturing a drug for the treatment of aninfectious disease. Advantageously, the infectious disease is selectedfrom those induced by viruses (human immunodeficiency virus or HIV,hepatitis B or C virus (HBV, HCV), Epstein-Barr virus or EBV,cytomegalovirus or CMV, enterovirus, influenza with Influenza virus A, Band C, syncytial respiratory virus or SRV, or HTLV), bacteria and/ortheir toxins (tetanus, diphtheria, pneumococci, meningococci,staphylococci including methicilin resistant forms, Klebsiellas,Shigellas, pseudomonas aeruginosa, enterobacteria or antiotic resistingpathologies including nosocomial diseases), parasites (paludism,leishmaniosis, trypanosomiasis) as well as emerging diseases, forexample Chikungunya, bird flu, severe acute respiratory syndrome virusor SARS, viruses responsible for haemorrhagic fevers such as Ebola orDengue fever or West Nile virus, and those related to bio-terrorism,such as Anthrax, Botulism, Plague, smallpox and poxvirus, Tularaemia,haemorrhagic fever agents, brucellosis, Staphylococcus B Enterotoxins,diphtheric toxin or viral Encephalitis.

DETAILED DESCRIPTION Kit of Parts

The term “kit of parts” designates a drug combination the elementaryconstituents of which form a functional unit due to their commonindication. More specifically, the kit of parts of the invention is adrug combination containing, as active substance, an effector cell whichexpresses CD16 on its surface and a monoclonal antibody in which theaffinity of the monoclonal antibody Fc region for CD16 is greater thanthe affinity of the polyclonal immunoglobulins Fc region for CD16, forsimultaneous, separate or sequential use, for the treatment of malignantpathologies, auto-immune or infectious diseases.

The monoclonal antibody and the effector cell, which expresses CD16 onits surface, together form a composition in the form of an unitary kitof parts, the constituents of which are available for simultaneous,separate or staggered over time application. The kit of parts of theinvention may also be in the form of a mixture. The monoclonal antibodyand the effector cell which expresses CD16 form a functional unit due toa novel common effect and thus a common indication.

Effector Cell which Expressing the FcγRIII Receptor (CD16) on itsSurface

By “Effector cell expressing the CD16 receptor on its surface”, it ismeant any cell capable of an effector activity (in particular cytotoxicactivity by ADCC, phagocytosis or, in another field, of antigenicpresentation and humoral response properties) following cellularactivation induced by the binding of an immune complex formed by theassociation of an antibody with the antigen it is specific for theFcγRIII or CD16 membraneous receptor. These cells necessarily expressCD16 on their surface. Advantageously, such a cell may be a cell derivedfrom monocyte or a monocyte precursor derived-cell which expresses CD16on its surface, a monocyte CD16+, a macrophage, a dendritic cell, thislist not being limited.

Therefore, this list may also extend to Natural Killer (NK) and PBMC(Peripheral Blood Mononuclear Cell) cells. By “NK cells” it is meantlarge granulosar lymphocytes capable of a spontaneous cytotoxic activitywithout previous immunisation. By “PBMC” it is meant any mononucleatedcell of the peripheral blood (monocytes and lymphocytes), and whichexpresses CD16 on its surface. Such cells are thus capable of inducingADCC activity in the presence of the monoclonal antibodies of theinvention, due to the binding between the monoclonal antibodies Fcregion and the CD16 receptor expressed by the cell. The effector cell ispreferably a macrophage. The CD16+ monocyte (i.e. expressing CD16 on itssurface) is a monocyte sub-population expressing CD16 on its membranesurface. CD16+ monocytes are capable of phagocyting and inducing ADCCactivity.

The macrophage is one of the main players of inherent immunity andparticipates to the adaptive immunity. It comes from the differentiationof monocytes. By way of example, macrophages may be derived from acellular suspension strongly enriched in monocytes comprising a culturestep in a suitable culture medium (RPMI® medium for Roswell ParkMemorial Institute) containing M-CSF (Monocyte-Colony StimulatingFactor) or GM-CSF (Granulocyte Macrophage-Colony Stimulating Factor) toinduce differentiation of monocytes into macrophages. The latter may begenerated, for example, in six to seven days of culture.

It is also possible to produce macrophages from a composition enrichedwith blood cells obtained by cytapheresis carried out on a healthyindividual, and by conducting a step for culturing monocytes in aculture medium containing M-CSF (Monocyte Colony Stimulating Factor) orGM-CSF (Granulocytes Macrophages Colony Stimulating Factor). Optionally,this culture step may advantageously be preceded firstly by a separationstep of, firstly, mononuclear cells, and, in an other hand, red bloodcells, granulocytes and part of the platelets contained in the bloodderived composition obtained by cytapheresis, and by an eliminationstep, by washing of a part of the blood platelets and anticoagulantsremaining than the preceding step. The abovementioned enriching step ofthe cellular suspension in monocytes is generally achieved bycentrifugating the medium containing the monocytes on a densitygradient, especially on a solution having a density of about 1.0 toabout 1.3 g/ml, such as a solution of Ficoll Paque type (Pharmacia)having a density of 1.077 g/ml.

Optionally, a composition containing macrophages, and/or dendriticcells, and/or NK cells may be obtained starting from a blood derivedcomposition of human origin, and enriched in blood cells, and, moreparticularly, in white blood cells such as monocytes, or precursorsthereof, especially a blood derived composition such as those obtainedby cytapheresis, said process comprising the following steps:

Advantageously, diluting of said blood derived composition, especiallyin about 2 to 3 times the volume thereof, by means of a suitablephysiological solution,

washing said blood derived composition, advantageously by simplecentrifugation and washing of the pellet resulting from theabove-mentioned centrifugation, after recovery of the pellet, insuitable physiological washing solution, especially in a pocket (oftransfer pocket type), by exerting pressure for example on said pocket,the washing solution then being eliminated to another pocket or otherreceptacle, to recover a composition deprived of any possibleanticoagulants and of any diverse residues, and impoverished inplatelets,

if required, repeating the abovementioned washing step, especiallybetween 1 and 2 times,

culturing the cells contained in the blood derived composition obtainedafter the abovementioned washing step, by placing the latter in anappropriate culture medium, especially in an advantageously hydrophobicpocket, for about 6 to about 10 days (especially about 6 to 7 days),

this culture step being:

preceded by an elimination step of all or part of the constituents otherthan the monocytes, or their precursors, which are likely to be presentin the starting composition, especially platelets, red blood cells,granulocytes and lymphocytes, by placing the blood derived compositionobtained after the washing step preceding the culture step in contactwith antibodies directed against all or part of the abovementionedconstituents, and recovering the solution containing the monocytes, ortheir precursors, while all or part of the abovementioned constituentsremain fixed to the antibodies, and/or followed by an elimination stepof all or part of the constituents other than the macrophages bycontacting the blood derived composition obtained after the culture stepwith the antibodies such as described hereinabove, and recovering thesolution containing the macrophages, while all or part of theabovementioned constituents remain fixed to the antibodies,

and/or followed by a purification step, especially by elutriation, inwhich the macrophages are physically separated from the otherconstituents of the composition obtained after the culture step,especially from the platelets, red blood cells and lymphocytes.

More generally, any other process for obtaining macrophages, resultingin the expression of CD16 on their surface, is also applicable to theinvention. In addition, by “macrophage”, in the present invention, it ismeant any cell obtained from monocytes and which is differentiatedaccording to a well determined protocol, thus resulting in cellsexpressing the following membrane markers: CD14+, CD16+, CD32+, CD64+,CD11b+. In particular, the percentage of CD16+ cells is of at least 20%,preferably 50%, or 70% or is comprised between 70 and 100%.

Monoclonal Antibodies

For the purposes of the invention, the expressions “monoclonal antibody”or “composition of monoclonal antibody” refer to a preparation ofantibody molecules originating from a cellular clone and having anidentical and single specificity. A molecule of immunoglobulin iscomposed of 4 polypeptides: 2 identical heavy chains (H, Heavy) of 50kDa each and 2 identical light chains (L, Light) of 25 kDa each. Thelight chain is composed of 2 domains, a variable domain V and a constantdomain C, folded back independently of one another in space, called VLand CL. The heavy chain also includes a V domain noted as VH and 3 or 4C domains noted as CH1 to CH4. Each domain comprises about 110 aminoacids and is structured comparably. The 2 heavy chains are linked bydisulfide bridges and each heavy chain is linked to a light chain by adisulfide bridge also.

The region determining the specificity of the antibody for the antigenis borne by the variable parts, while the constant parts may interactwith the Fc receptors of the effector cells or with molecules such asproteins of the complement to cause different functional properties. Asregards the variable regions of the heavy and light chains, it isobserved that the variability in sequence is not distributed equally. Ineffect, the variable regions are constituted both by regions onlyslightly variable known as “framework” (FR), numbering 4 (FR 1 to FR4)and also by regions in which variability is extreme: these are“hypervariable” regions, or CDR (for Complementarity DeterminingRegions), totalling 3 (CDR1 to CDR3).

Advantageously, the antibody according to the invention is a chimeric,humanised or human antibody. The antibody according to the invention ispreferably chimeric. By “Chimeric antibody”, it is meant an antibody,the variable regions of the light chains and the heavy chains of whichbelong to a different species from that the constant regions of thelight chains and the heavy chains belong to. Therefore, the antibodyaccording to the invention also has variable regions of murine, rat,rabbit, monkey, goat, or human origine and constant regions which belongto a species different from the species where the antibody was produced.In this respect, all the families and species of mammals are likely tobe used, and in particular human being, monkey, rats and mice, swine,bovines, equines, felines, canines, for example, as well as birds. Evenmore preferably, the constant regions of each of the light chains andeach of the heavy chains of the antibody according to the invention arehuman constant regions. This preferred embodiment of the inventionallows to decrease the immunogenicity of the antibody in humans andthereby to improve its efficiency during its therapeutic administrationin human.

In a preferred embodiment of the invention, the constant region of eachof the light chains of the antibody according to the invention is ofκ-type. Any allotype is suitable for achieving the invention, forexample Km(1), Km(1, 2), Km(1, 2, 3) or Km(3). In another embodiment ofthe invention, the constant region of each of the light chains of theantibody according to the invention is of λ-type.

In a particular aspect of the invention, and especially when theconstant regions of each of the light chains and of each of the heavychains of the antibody according to the invention are human regions, theconstant region of each of the heavy chains of the antibody is ofγ-type. According to this variant, the constant region of each of theheavy chains of the antibody may be of γ1-type, of γ2-type, of γ3-type,these three types of constant regions having the particular feature offixing the human complement, or even of γ4-type. The antibodies, theheavy chains of which have a γ type constant region belong to the classof IgG. The G-type immunoglobulins (IgG) are heterodimers constituted by2 heavy chains and 2 light chains, linked to one another by disulfidebridges. Each chain is constituted, in the N-terminal position, by avariable region or domain (coded by the rearranged genes V-J for thelight chain and V-D-J for the heavy chain) which is specific for theantigen against which the antibody is directed, and in the C-terminalposition, of a constant region, constituted by a single CL domain forthe light chain or of 3 domains (CH1, CH2 and CH3) for the heavy chain.The association of the variable domains and the CH1 and CL domains ofthe heavy and light chains forms the Fab fragments which are connectedto the Fc region by a very flexible hinge region allowing each Fab to befixed to its antigenic target while the Fc region, which mediates theeffector properties of the antibody, remains accessible to the effectormolecules such as the FcγR and the C1q receptors. The Fc region,constituted by 2 globular domains Cγ₂ and Cγ₃, is glycosylated at thelevel of the Cγ₂ domain with the presence, on each of the 2 chains, of abiantenna N-glycan, linked to asparagine 297. The constant region ofeach of the heavy chains of the antibody is preferably of γ1-type, sincesuch an antibody shows the capacity to engender ADCC activity(Antibody-Dependent Cellular Cytotoxicity) in the greatest number of(human) individuals. In this respect, any allotype is suitable forachieving the invention, for example G1m(3), G1m (1, 2, 17), G1m(1, 17)or G1m(1,3).

The chimeric antibodies according to the invention may be constructedusing standard recombinant DNA techniques, well known to those skilledin the art, and more particularly using the construction techniques ofchimeric antibodies described for example in Morrison et al., Proc.Natl. Acad. Sci. U.S.A., 81: 6851-55 (1984), where DNA recombinanttechnology is used for replacing the constant region of a heavy chainand/or the constant region of a light chain of an antibody originatingfrom a non-human mammal with the corresponding regions of a humanimmunoglobulin. Such antibodies and their preparation method have alsobeen described in patent EP 173 494, in the document Neuberger, M. S. etal., Nature 1985 Mar. 21-27; 314(6008): 268-70., as well as in thedocument EP 125 023, for example. Methods for generating chimericantibodies are widely available for those skilled in the art. Forexample, the heavy and light chains of the antibodies may be expressedseparately using a vector for each chain, or they may be integrated intoa single vector.

An expression vector is a nucleic acid molecule in which the nucleicacid sequence coding for the variable domain of each of the heavy orlight chains of the antibody and/or the nucleic acid sequence,preferably human, coding for the constant region of each of the heavy orlight chains of the antibody have been inserted, so as to introduce andkeep them in a host cell. It allows expression of these foreign nucleicacid fragments in the host cell since it has indispendable sequences(promoter, polyadenylation sequence) to this expression. The vector maybe for example a plasmid, an adenovirus, a retrovirus or abacteriophage, and the host cell may be any mammalian cell, for exampleSP2/0, YB2/0, IR983F, Namalwa human myeloma, PERC6, CHO lines,especially CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHOdhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NS0,SP2/0-Ag 14 and P3X63Ag8.653.

For constructing expression vectors for the chimeric antibodiesaccording to the invention, synthetic signal sequences and suitablerestriction sites may be fused to the variable regions during PCRamplification reactions (Polymerase Chain Reaction). The variableregions are then combined with the constant regions of an antibody,preferably a human IgG1. The genes thus constructed are cloned under thecontrol of a promoter (for example the RSV promoter) and upstream of apolyadenylation site, using either two separate vectors (one for eachchain) or a single vector. The vector(s) is (are) also provided withselection genes known to those skilled in the art, such as for examplethe dhfr gene, the neomycin resistance gene. The chimeric antibodiesaccording to the invention may be produced by co-transfecting or singletransfecting the light chain expression vector of the heavy chainexpression vector or the single vector in a host cell through the use ofa method well known to those skilled in the art (for exampleco-precipitation with calcium phosphate, electroporation,micro-injection, etc.).

By Humanised antibody, it is meant to refer to an antibody containingCDRs regions derived from a non-human antibody, the other parts of theantibody molecule being derived from one (or more) human antibodies.Such antibodies may be prepared according to CDR grafting methods(“CDR-grafting”) well known to those skilled in the art (U.S. Pat. No.5,225,539, U.S. Pat. No. 6,180,370; Jones et al., Nature 321(6069):522-5. (1986); Verhoeyen et al., Bioessays 8(2): 74-8 (1988); Riechmannet al., Nature 332: 323-7 (1988); Queen C. et al. Proc. Natl. Acad. Sci.U.S.A. 86(24):10029-33 (1989); Lewis A. P. and Crowe J. S., Gene 101(2):297-302 (1991); Daugherty B L et al. Nucleic Acids Res. 19(9): 2471-6(1991); Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992);Singer et al., J. Immunol. 150 (7): 2844-57 (1993); Presta et al., J.Immunol., 151: 2623 (1993)). The choice of variable human domains to begrafted for producing humanised antibodies is important for reducing theantibody immunogenicity without altering its affinity for its target. Ina production method for a humanised antibody, the sequence of thevariable domain of a murine antibody is compared to a sequence libraryof known variable human regions and the closest variable human sequenceto the murine sequence is retained as FR region (“framework”) of thehumanised antibody [Riechmann et al., Nature 332: 323-7 (1988); Queen C.et al., Proc. Natl. Acad. Sci. USA 86(24): 10029-33 (1989); Sims et al.,J. Immunol., 151: 2296 (1993)]. Another selection method of human FRregions is the comparing the sequence of each murine FR sequencesub-region (FR1, FR2, FR3 and FR4) with a known human FR sequenceslibrary, such as to select, for each FR region, the closest human FRsequence to the murine sequence [US patent 2003/0040606; Singer et al.,J. Immunol. 150 (7): 2844-57 (1993); Sato K. and et al, Mol. Immunol.31(5): 371-81 (1994); Leung S. O. et al., Mol. Immunol. 32 (17-18):1413-27 (1995)]. Another method uses a particular FR region which isderived from a consensus sequence of a particular sub-group of humanantibodies heavy or light chain [Sato K. and et al, Mol. Immunol. 31(5):371-81 (1994)]. The CDR graft is completed in the majority of cases bymuting some key residues localised in human FRs in order to conserve agood affinity of the humanised antibody for its target [Holmes M. A. andFoote J., J. Immunol. 158(5): 2192-201 (1997)].

The humanised antibodies according to the invention are preferred fortheir use in in vitro diagnostic methods, or in vivo prophylactic and/ortherapeutic treatment methods. The thus chimerised or humanised antibodyaccording to the invention has the advantage of being better toleratedby the human organism, and at least as efficient as the originalantibody. In a particularly advantageous way, the thus chimerised orhumanised antibody is twice as cytotoxic as the corresponding nativeantibody. In an even more advantageous way, the thus chimerised orhumanised antibody is 10 times, or even 100 times or preferably morethan 500 times more cytotoxic than the corresponding native antibody.

By human antibody, it is meant to refer to an antibody each region ofwhich is derived from a human antibody. These antibodies may be derivedfrom transgenic mice carrying human antibodies genes or from human cells[Jakobovits et al., Curr Opin Biotechnol. October; 6(5): 561-6 (1995);Lonberg N. and D. Huszar. Internal Review of Immunology 13: 65-93(1995); Tomizuka K. et al., Proc. Natl. Acad, Sci. USA 97(2): 722-727(2000)]. The humanised or human chimeric antibodies of the invention arepreferably produced by way of recombinant DNA techniques known to thoseskilled in the art. The monoclonal antibodies of the invention maypreferably be produced by an isolated cell, for example selected fromSP2/0, YB2/0, IR983F, Namalwa human myeloma, PERC6, the CHO lines,especially CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHOdhfr-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NS0,SP2/0-Ag 14 and P3X63Ag8.653, this list not being limited.

The monoclonal antibodies of the invention may also be produced by wayof a transgenic animal. Transgenesis is a molecular genetic technique bywhich exogenic DNA is introduced into the genome of a multicellularorganism and is transmitted to the progeny thereof. This transmission toprogeny imposes stable integration of said DNA in the genome of theembryo, at an early stage of development. For example, one of thetransgenesis techniques likely to be used within the scope of theinvention consists in micro-injecting naked DNA into the pronucleus in afertilized mammal ovocyte or into embryonic stem cells, which leads, ina certain number of cases, to the integration of part of microinjectedDNA molecules into the host genome. Other techniques may be used fortransgenesis, and especially techniques for introducing exogenic DNAinto a living cell, which are well known to those skilled in the art,especially electroporation, transfection by means of calcium phosphateprecipitates, modified liposomes or lipids such as Lipofectamine® (INVITROGEN).

The monoclonal antibody of the invention is preferably produced by thetransgenic animal in its milk. In this way, the gene coding for theprotein of interest is associated with gene-regulating elementsexpressed specifically in milk (for example the promoter of the WAPgene, whey acidic protein). The resulting expression vector ismicro-injected under microscope into mammalian embryos at theunicellular stage. The embryos are then transferred to receivingfemales.

For example, after one month of gestation, the first mammals which hadintegrated the transgene (F0) into their genome are being born and areidentified by ear biopsy PCR analysis. They will be used as founders togive birth to the second generation of transgenic mammals. The foundersare selected for their efficiency to produce the protein of interest intheir milk and for generating the second generation of transgenicrabbits (F1).

The F1 progeny is identified by ear biopsy followed by PCR analysis. Thesexually mature F1 females are then inseminated with sperm fromnon-transgenic males. The milk is harvested mechanically and therecombinant protein is characterised such as to select the best line forlarge-scale production and for developing the purification strategy(GLP, pre-GMP, GMP).

In parallel, the sperm of F1 transgenic males—Master Sperm Bank, MSB—isharvested and cryo-conserved in liquid nitrogen, followingrecommendations of the FDA and of European instances. This sperm will beused for artificially inseminating non-transgenic females to generatethe second progeny (F2). The sperm of F2 transgenic males—WorkingSpermBank, WSB—is harvested and over 15 to 20 years will serve togenerate F3 transgenic females which will produce industrial quantitiesof the monoclonal antibody in their milk. This type of technique isdescribed for example in patent EP 0 527 063.

Fc Gamma Receptors

CD16, also called type III Fc gamma receptor (FcγRIII), is a receptorpresent on numerous immune system cells. Together with CD32 (FcγRII) andCD64 (FcγRI), CD16 is a specific receptor for constant (Fc) fragmentsIgG antibodies heavy chains. Binding of an immune complex, via the Fc ofIgG, to these CD16, CD32 and CD64 receptor which are present on theimmune system effector cells activates the latter and especially immunecomplex phagocytosis.

The effector cells of the invention express on their cellular membrane 3types of Fc receptors: CD64, CD32 and CD16. The CD16 receptor istraditionally called “low-affinity receptor”, and is expressedconstitutionally on the PMNs (polymorphonuclear neutrophils), asub-population of monocytes, on macrophages, dendritic cells and NaturalKiller cells (NK cells). CD16 participates in multiple effectorfunctions, for example phagocytosis, opsonisation of particles or ofimmune complexes, and ADCC activity.

The monoclonal antibody of the kit of parts of the invention has an Fcregion exhibiting strong affinity for the Fc receptors which are presenton the effector cells of the invention, and in particular for CD16. Theinvention describes the synergy between the Fc region of the monoclonalantibodies of the invention and CD16 of the effector cells of the kit ofparts. This affinity is such that the addition of human polyvalentplasmatic IgG (important constituent of peripheral blood) in the mediumcontaining antibodies and effector cells has no or little influence onADCC activity generated by the association between the monoclonalantibody and the effector cells. This is due to the fact that theaffinity of the Fc region of the monoclonal antibody for CD16 is greaterthan that of the human IgG which are present in physiologicalconditions. As a consequence, ADCC activity observed in vitro will notbe diminished in vivo following the absence of displacement of theantibody of interest by seric IgG. In effect, plasma and serum containstrong concentrations of polyvalent immunoglobulins (also calledpolyvalent plasmatic IgG or polyclonal IgG or seric IgG). The monoclonalantibody of the kit of parts induces activation of the effector cellsvia the Fc receptors the CD16 and the CD64 of which lead to cellularlysis by ADCC or phagocytosis. It is now commonly admitted thatpolyvalent plasmatic IgG inhibit the lysis mechanism of the effectorcells via CD64, the latter being saturated in the presence of polyvalentIgG.

The applicant has shown that the association in a kit of parts of amonoclonal antibody the Fc region of which has a greater affinity forCD16 than that of the plasmatic fractions isolated IgG surprisinglyinduces an ADCC activity which is not inhibited by the addition ofplasmatic IgG in vitro, thus making feasible to conserve the therapeuticactivity in vivo. This in vivo therapeutic activity corresponds to thelysis of tumour cells, of cells infected by pathogenic agents or ofcells producing auto-antibodies. Therefore, advantageously, themonoclonal antibody is not displaced by polyvalent IgG in the case ofthe addition of human plasmatic IgG.

Due to the strong affinity of the antibody Fc region for CD16, themonoclonal antibody binds to the effector cells, and this binding is notdisplaced by the human polyvalent plasmatic IgG, even at strong serumconcentrations. As a consequence, the kit of parts of the inventionenables an optimal lysis of the target cells even at low concentrationsof the monoclonal antibody. Advantageously, the concentration of themonoclonal antibody of the kit of parts is less than the concentrationof an antibody with the same specificity, traditionally used inmonotherapy for treating malignant pathologies, auto-immune orinfectious diseases.

In a particular embodiment of the invention, the Fc region of themonoclonal antibody of the invention has an association constant withthe CD16 of at least 2.10⁶ M⁻¹. Advantageously, the association constantof the antibody of the invention is measured according to the methoddescribed in the document Maenaka et al. (Katsumi Maena, P. Anton vander Merwe, David I. Stuart, E. Yvonne Jones, and Peter Sondermann; TheHuman Low Affinity Fcy receptors IIa, Iib, and III bind IgG with FastKinetics and Distinct Thermodynamic Properties. J. Biol. Chem., Vol.276, Issue 48, 44898-44904, Nov. 30, 2001).

In a preferred embodiment of the invention, the monoclonal antibodyconcentration in the kit of parts is preferably less than 1 mg/200millions of cells. The use of the invention, hereinabove called a kit ofparts, is considered in pathologies or after injection. Theeffector/target ratio is not necessarily high, i.e. less than 10, oreven 1 or 0.1.

In a particular aspect of the invention, the monoclonal antibody bindsof the effector cell CD16 with an affinity of at least 2.10⁶ M⁻¹. Forexample, the monoclonal antibody of the invention may be prepared bymeans of the process described in patent application WO 01/77181. Thisprocess for preparating a monoclonal antibody capable of activating theCD16 expressing effector cells comprises the following steps:

a) purificating monoclonal antibodies obtained from various clonesoriginating from cell lines selected from hybridomes, especiallyheterohybridomes and animal or human cell lines transfected by means ofa vector comprising the gene coding for said antibody;

b) adding each antibody obtained in step a) in a distinct reactionalmixture comprising:

i. the target cells of said antibody,

ii. effector cells comprising FcγRIII expressing cells

iii. polyvalent IgG,

c) determinating the lysis percentage of target cells and selectingmonoclonal antibodies which activate the effector cells causing asignificant lysis of the target cells (ADCC activity depending fromFcγRIII).

For each antigenic specificity, the monoclonal antibody of the inventionis in reality a composition containing monoclonal antibodies, all ofthem being identical at the level of their primary structure since theyall originate from the same cellular clone. However, all antibodies of amonoclonal antibodies composition do not exhibit the same glycannicprofile. Human and animal antibodies have a N-bond oligosaccharide onthe CH2 domain of each of their heavy chains. The binding site of thisoligosaccharide is, for G immunoglobulins, asparagine 297 (Asn 297according to Kabat). This asparagine residue is also called “Fcγglycosylation”.

The extremity of the oligosaccharide chain bound to Asn 297 is called“reductor extremity”, whereas the opposite extremity is called “nonreductor extremity”. In the Fc region of the IgG antibodies, there aretwo Fcγ glycosylation sites; therefore two oligosaccharide chains arebound to each antibody molecule.

Therefore, in a monoclonal antibodies composition, the oligosaccharidechains have varied structures, depending from the glycosylationconferred by the productive cell line. However, these chains have acommon base structure:

This base structure, common to all monoclonal antibodies, may furthercomprise the following sugars: N-acetylglucosamine (GIcNAc), fucose(fuc) and galactose (gal). The principal glycosylated forms ofN-oligosaccharides are shown below:

Since each oligosaccharide chain may include one or more of thesesugars, and may thus present itself in the herein above illustrated G0,G0F, G1 or G1F form, there is, in a monoclonal antibodies composition, amultitude of combinations of oligosaccharides conferring to themonoclonal antibodies composition a ratio in each of these sugars whichmay be different from one antibody composition to the other. Therefore,clones originating from the same cell line may produce antibodycompositions the glycannic compositions of which may vary. Therefore, ithas been shown, surprisingly, by the applicant that the monoclonalantibodies compositions in which the rate of fucose is less than 65%have a strong affinity for CD16. More particularly, this type ofmonoclonal antibodies composition has an affinity of their region forCD16 which is higher than that of the polyvalent IgG for CD16. Inaddition, the monoclonal antibodies of the composition are not displacedby seric Ig.

A method for preparating of such monoclonal antibodies compositions isgiven for example in patent application WO 01/77181. In an advantageousembodiment of the invention, the monoclonal antibodies composition isproduced by a cell having low enzymatic activity allowing the additionof fucose to N-acetylglucosamine of the reducing extremity, such anenzyme being preferably fucosyltransferase. In another embodiment of theinvention, it is possible to have act on the monoclonal antibodiescomposition an enzyme, for example fucosidase, so as to obtain amonoclonal antibodies composition comprising such a rate of fucose. In apreferred embodiment of the invention, the monoclonal antibodiescomposition is produced in YB2/0 (ATCC CRL-1662).

By way of advantage, the monoclonal antibody of the kit of parts of theinvention is directed against the 5C5 antigen (tumorous antigenexpressed by the cells of renal carcinomas), BCR (B Cell Receptor), anidiotype such as that of anti-FVIII inhibitors antibodies, TCR (T CellReceptor), CD2, CD3, CD4, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD45, CD30, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66(a,b,c,d), CD74, CD80, CD86, CD126, CD138, CD154, MUC1 (Mucine 1), MUC2(Mucine 2), MUC3 (Mucine 3), MUC4 (Mucine 4), MUC16 (Mucine 16), HM1.24(specific antigen for plasmocytes which is overexpressed in multiplemyelomas), tenascin (protein of the extra-cellular matrix), GGT(gamma-glutamyltranspeptidase), VEGF (Vascular Endothelial GrowthFactor), EGFR (Endothelial Growth Factor receptor), CEA(carcinoembryonic antigen), CSAp (colon-specific antigen-p), ILGF(Insulin-Like Growth factor), placental growth factor, Her2/neu,carbonic anhydrase IX, IL-6, S100 proteins (multigenic family ofproteins linking to calcium), MART-1 (tumorous differentiation antigenassociated with melanoma), TRP-1 (tyrosinase-related protein 1), TRP-2(tyrosinase-related protein 2), gp100 (glycoprotein 100 kDa), amyloidproteins, rhesus D antigen, MHC molecules of class I and II such asHLA-DR, an antigen resulting from the expression of mutated genesespecially oncogenes or tumour-suppress genes, an antigen derived fromoncogenic viruses which have expressed by certain tumours, a ubiquitousantigen overexpressed in some tumours and slightly expressed in somenormal tissues, such as for example the type II receptor of theMüllerian hormone, a glycosylated or non-glycosylated protein, aphospholipid, a molecule of the self or of the non-self expressed orexposed on the membrane by infected cells such as phosphatidylserine,and a protein expressed or secreted by a pathogenic agent (bacterialtoxin, protein complexes of the bacterial or parasitic wall, viralenvelope glycoproteins, for example from HIV virus, HBV, HCV, RSV,etc.), this list not being limited.

The antibody of the invention is preferably directed against the CD20.The CD20 antigen is a hydrophobic transmembrane protein with a molecularweight of 35-37 kDa which is present on the surface of mature Blymphocytes (Valentine et al. 1987, Proc Natl Acad Sci U.S.A. 84(22):8085-9; Valentine et al. 1989, J. Biol. Chem. 264(19): 11282-11287). Itis expressed during the development of B lymphocytes from the earlypre-B stage until differentiation in plasmocyte, a stage where thisexpression disappears. The CD20 antigen is present both on normal Blymphocytes and on malignant B cells. More particularly, the CD20antigen is expressed on the most of B phenotype lymphomas (80% oflymphomas): it is expressed for example on more than 90% of lymphocytesB non-Hodgkin's lymphomas (NHL), and on more than 95% of B type ChronicLymphoid Leukemias (LLC-B). The CD20 antigen is not expressed on thehaematopoietic stem cells or on the plasmocytes.

The function of CD20 is not yet fully clarified, though it could act asa calcic channel and intervene in the regulation of the first steps ofdifferentiation (Golay et al. 1985, J. Immunol.; 135(6): 3795-801) andof proliferation (Tedder et al. 1986, Eur J. Immunol. 1986 August;16(8): 881-7) of B lymphocytes. In a preferred embodiment of theinvention, the composition of anti-CD20 antibodies is produced by YB2/0and has a fucose rate of less than 65%. In a particular embodiment ofthe invention, such an antibody, and its production process, aredescribed in patent application WO2006/064121.

Advantageously, the amino acid sequence of the heavy chain of such anantibody is the sequence set forth in SEQ ID NO: 1. Advantageously, theaminoacid sequence of the light-chain of such an antibody is thesequence set forth in SEQ ID NO: 2 or 3.

In brief, this antibody may be obtained, in accordance with the teachingof patent application WO2006/064121, by means of YB2/0 cell transfectionby vectors allowing the expression of the hereinabove described lightchain and heavy chain. In a preferred embodiment of the invention, thecomposition of monoclonal anti-CD20 antibody has a fucose rate of lessthan 65%, and comprised preferably between 20 and 40%, or afucose/galactose ratio of less than 0.6. In a preferred embodiment ofthe invention, the monoclonal antibody of the kit of parts is producedby the R509 clone, deposited to the CNCM under accession number CNCMI-3314. In another preferred embodiment of the invention, the monoclonalantibody of the kit of parts is produced by the R603 clone, deposited tothe CNCM under accession number CNCM I-3529.

The applicant has shown that the kit of parts of the invention isefficient for treating LLC-B, since malignant cells from patients withLLC-B were lysed ex vivo, and even at a ratio less than or equal to 10,or even 5 or even 2 E:T, at low antibody concentrations, including inthe presence of human serum. The kit of parts of the invention thusallows optimal lysis of the target recognised by the variable regions ofthe antibody, due to the physical interactions (binding) between theeffector cells and the Fc region of the antibodies, which issufficiently strong not to be displaced by the polyvalent IgG.Advantageously, the concentration of monoclonal antibody contained inthe kit of parts of the invention for treating LLC-B is of less than 375mg/m².

Because of its advantages as regards of low toxicity, specificity andreduced dose, the kit of parts comprising the anti-CD20 antibodies maybe administered for treating the following pathologies: malignantpathologies with a lymphoproliferative syndrome of CD20 positive Blymphocytes with for example type B NHL or acute or chronic lymphoid Bleukemias, auto-immune and/or inflammatory diseases such as organ graftsrejection, graft versus host disease, rheumatoid polyarthritis,disseminated lupus erythematosus, sclerodermia, primitive Sjögren'ssyndrome (or Gougerot-Sjögren's Syndrome), auto-immune polyneuropathiessuch as multiple sclerosis, type I diabetes, auto-immune hepatitis,ankylosing spondylarthritis, Reiter's syndrome, gout arthritis, coeliacdisease, Crohn's disease, Hashimoto's thyroiditis, Addison's disease,auto-immune hepatitis, Basedow's disease, ulcerative colitis, vasculitissuch as systemic vasculitis associated with ANCA (anti-neutrophilcytoplasmic antibody), auto-immune cytopenias and other haematologicalcomplications in adults and children, such as acute or chronicauto-immune thrombopenias, auto-immune haemolytic anaemias, haemolyticdisease of the newborn (HDN), cold agglutinin disease, thrombocytopenicthrombotic purpura and acquired auto-immune haemophilia; Goodpasture'ssyndrome, extra-membraneous nephropathies, auto-immune bullous skindisorders, refractory myasthenia, mixed cryoglobulinemias, psoriasis,juvenile chronic arthritis, inflammatory myositis, dermatomyositis andchildren systemic auto-immune diseases including antiphospholipidssyndrome, this list not being limited.

Advantageously, the kit of parts of the invention is an injectablesolution. This injectable solution is advantageously in the form of alocally or systemically injectable solution. In a particular embodiment,6 administrations are done to the patient. One administration is doneper day or every two days over a week, then once per week over one monthor two, one administration three times/month, the cure being renewableseveral times.

In a complementary embodiment, the effector cells are administered at adose comprised between 10⁴ and 10⁹ effector cells per injection. Inanother complementary embodiment, the antibodies of the invention areadministered at a dose comprised between 1 and 500 mg of antibodies perinjection.

In another particular embodiment of the invention, the effector cellsare administered repeatedly up to 10 times, the time interval betweeneach administration being comprised between 2 days and 12 months. Inanother particular embodiment of the invention, the monoclonal antibodyis administered repeatedly up to 10 times, the time interval betweeneach administration being comprised between 2 days and 12 months. Inanother embodiment of the invention, the monoclonal antibody and theeffector cells are administered simultaneously.

In another embodiment of the invention, the monoclonal antibody and theeffector cells are administered sequentially, the monoclonal antibodybeing administered before the effector cells. In another embodiment ofthe invention, the monoclonal antibody and the effector cells areadministered sequentially, the monoclonal antibody being administeredafter the effector cells. Another object of the invention is apharmaceutical composition comprising the kit of parts of the invention.Another object of the invention relates to the use of the kit of partsof the invention for preparing a drug for treating malignant,auto-immune and infectious pathologies.

This drug or pharmaceutical composition advantageously comprises anexcipient and/or a pharmaceutically acceptable vehicle. The excipientmay be any solution, such as a saline, physiological, isotonic, bufferedsolution, etc., as well as any suspension, gel, powder, etc., compatiblewith pharmaceutical usage and known to those skilled in the art. Thecompositions according to the invention may also contain one or moreagents or vehicles selected from dispersants, solubilisers, stabilisers,surfactants, preservatives, etc. Also, the compositions according to theinvention may comprise other agents or active ingredients.

Another object of the invention is the use of the kit of parts of theinvention for manufacturing a drug. Another object of the invention isthe use of the kit of parts of the invention for manufacturing a drugfor treating a malignant pathology.

Advantageously, this malignant pathology is selected from solid tumoursand malignant haemopathies. Solid tumours are selected from melanomas,carcinomas, sarcomas, gliomas and skin cancers. Carcinomas are selectedin the group constituted by kidneys, breast, oral cavity, lungs,gastro-intestinal tract, ovaries, prostate, uterus, bladder, pancreas,liver, gallbladder, skin and testicles carcinomas. Malignanthaemopathies are selected from lymphoproliferative, myeloproliferative,myelodysplasic syndromes and acute myeloid leukemias with for exampletype B NHL, acute or chronics lymphoid B leukemias, Burkitt's lymphoma,tricholeucocyte leukaemia, acute and chronic myeloid leukemias, Tlymphomas and leukemias, Hodgkin's lymphomas, Waldenström'smacroglobulinemia and multiple myelomas, this list not being limited.

Another object of the invention is the use of the kit of parts of theinvention for manufacturing a drug intended for treating an auto-immuneand/or inflammatory primitive or secondary condition, which is specificto organs or systemics and which is associated or not with pathogenicauto-antibodies, selected from organ grafts rejection, graft versus hostdisease, rheumatoid polyarthritis, disseminated lupus erythematosus,sclerodermia, primitive Sjögren's syndrome (or Gougerot-Sjögrensyndrome), auto-immune polyneuropathies such as multiple sclerosis, typeI diabetes, auto-immune hepatitis, ankylosing spondylarthritis, Reiter'ssyndrome, gout arthritis, coeliac disease, Crohn's disease, Hashimoto'sthyroiditis, Addison's disease, auto-immune hepatitis, Basedow'sdisease, ulcerative colitis, vasculitis such as systemic vasculitisassociated with ANCA (antineutrophil cytoplasmic antibody), auto-immunecytopenias and other haematological complications in adults andchildren, such as acute or chronic auto-immune thrombopenias,auto-immune haemolytic anaemias, haemolytic disease of the newborn(HDN), cold agglutinin disease, thrombocytopenic thrombotic purpura andacquired auto-immune haemophilia; Goodpasture's syndrome,extra-membraneous nephropathies, auto-immune bullous skin disorders,refractory myasthenia, mixed cryoglobulinemias, psoriasis, juvenilechronic arthritis, inflammatory myositis, dermatomyositis and childrensystemic auto-immune diseases including antiphospholipids syndrome, thislist not being limited.

Another object of the invention is the use of the kit of parts of theinvention for manufacturing a drug for treating an infectious disease.Advantageously, this infectious disease is selected from those inducedby virus (human immunodeficiency virus or HIV, hepatitis B or C virus(HBV, HCV), Epstein-Barr virus or EBV, cytomegalovirus or CMV,enterovirus, influenza with the A, B and C Influenza virus, respiratorysyncytial virus or RSV, or HTLV), bacteria and/or their toxins (tetanus,diphtheria, pneumococci, meningococci, staphylococci includingmethicilin resistant forms, Klebsiellas, Shigellas, pseudomonasaeruginosa, enterobacteria or antibiotics resistant pathologiesincluding nosocomial diseases), parasites (paludism, leishmaniosis,trypanosomiases) as well as emerging diseases, for example Chikungunya,bird flu, severe acute respiratory virus syndrome or SARS, virusesresponsible for haemorrhagic fevers such as Ebola or Dengue fever orWest Nile virus, and those related to bio-terrorism, such as Anthrax,Botulism, Plague, smallpox and poxvirus, Tularaemia, haemorrhagic feveragents, brucellosis, Staphylococcus B Enterotoxins, diphtheric toxin orviral Encephalitis, this list not being limited. Other aspects andadvantages of the invention will be described in the following exampleswhich must be considered as illustrative and do not limit the scope ofthe invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Binding study of anti-D R297 EMABling antibodies and of AD1antibody to CD16 (FcγRIII receptor) of macrophages through a competitiontest;

FIG. 2: Macrophage induced ADCC activity of EMABling R297 antibodies andof AD1 antibody in the presence of various concentrations of polyclonalimmunoglobulins (IVIg);

FIG. 3: Macrophage induced ADCC activity of EMABling R297 antibodies andof AD1 antibody in the presence of various concentrations ofimmunoglobulins (IVIg) and of anti-CD16 3G8 antibody at a concentrationof 6.25 μg/ml; and

FIG. 4: Phagocytosis of Rh+erytrhocytes by CD16+ macrophages induced bythe EMABling R297 antibody and the AD1 antibody in the presence ofvarious concentrations of immunoglobulins (IVIg).

EXAMPLES Example 1 Differentiation of Monocytes in Macrophages

Monocytes are isolated from peripheral blood by fractionating on Ficoland Percol density gradient, then culturing in an RPMI medium containing10% SVF and adding of M-CSF (Monocyte Colony Stimulating Factors) (50ng/ml). After 7 days, the obtained macrophages are of CD14+, CD16+,CD32+, CD64+, CD11b+, CD1a−, CD80−, CD83− phenotype. Therefore, theM-CSF differentiation allows expression of CD16 on the surface ofmacrophages.

Example 2 Interaction of Anti-D Antibodies with CD16 Expressed byMacrophages

The binding of the anti-D R297 antibody (also called “EMABling R297”) iscompared to that of the AD1 antibody. The anti-D R297 antibody isdescribed in the document WO 01/77181, and is produced according to theprocess described in this document. This antibody is produced in theYB2/0 cell (ATCC CRL-1662). Binding of the R297 antibody on macrophagesCD16 is compared to that of the AD1 antibody (described in the documentWO 01/77181, expressed by a heteromyeloma).

The displacement assay of the anti-CD16 antibody (producer clone 3G8)allows to measure the binding of monoclonal antibodies on the CD16receptor of these macrophages, irrespective of their specificity. Thepurified macrophages are incubated with variable concentrations (0 to 83μg/ml) of anti-D antibody (R297 or AD1) and with the anti-CD16 3G8antibody coupled to a fluorochrome (3G8-PE) at a determinedconcentration.

After washing, binding of the antibody 3G8-PE on the CD16 receptor ofthe macrophages is evaluated by flow cytometry. The antibodies havingthe capacity to bind themselves on CD16 enter into competition with thebinding of the 3G8 antibody and, consequently, induce a decrease in MFI(Mean Fluorescence Intensity). The results are expressed in fluorescenceaverages (MFI), as a function of the quantity of antibodies to beevaluated.

FIG. 1 shows that the R297 antibody binds very strongly on macrophagesCD16 when compared to the AD1 antibody. At the plateau, the EMABlingantibody induces a displacement which is at least 6 times greater thanthe AD1 antibody.

Example 3 Interaction of Anti-CD20 EMAB6 and EMAB603 Antibodies withCD16 Expressed by Macrophages

The binding of anti-CD20 EMAB603 antibodies (produced by the R603 clone,deposite to the CNCM under the number I-3529) and EMAB6 (produced by theR509 clone, deposited to the CNCM under the number I-3314) onmacrophages CD16 is compared to that of Rituxan. The anti-CD20 EMAB6 andEMAB603 antibodies are produced according to the process described inpatent application WO2006/064121, in particular at pages 26-33. Also,the clones producing these antibodies are available at CNCM under theaccession numbers CNCM I-3314 and CNCM I-3529, respectively. Thedisplacement assay of the anti-CD16 antibody (producer clone 3G8)measures the binding of the monoclonal antibodies on the CD16 receptor,irrespective of their specificity.

The macrophages are incubated with variable concentrations (0 to 83μg/ml) of anti-CD20 antibody (EMAB6, EMAB603 or rituximab) and with theanti-CD16 3G8 antibody coupled to a fluorochrome (3G8-PE) at adetermined concentration. After washing, binding of the 3G8-PE antibodyon the CD16 receptor of the macrophages is evaluated by flow cytometry.The antibodies having the capacity to bind themselves on CD16 enter intocompetition with the binding of the 3G8 antibody, and consequentlyinduce a decrease in MFI (Mean Fluorescence Intensity). The results areexpressed in fluorescence averages (MFI), as a function of the quantityof antibodies to be evaluated.

The Imax values (maximal inhibition of 3G8 binding) and IC50 values(anti-CD20 antibody concentration required to induce a 3G8 bindinginhibition of 50% of Imax) are calculated using PRISM statisticalanalysis software.

Result: the interaction of EMABling R603 and EMAB6 antibodies onmacrophages CD16 is much greater than that obtained with Rituxan.Therefore, since this assay is performed in the absence of an antigenictarget, the anti-CD20 antibodies of the invention have the capacity tobe bind strongly on macrophages CD16.

Example 4 Anti-D/Erytrhocytes Rh+/Macrophages ADCC Activity. Role ofIVIg Polyvalent (Tegeline®)

The cytotoxic capacity of anti-D antibodies is studied by the ADCCtechnique. The anti-D antibodies, macrophages (differentiated monocytesin M-CSF) and Rhesus D+ erythrocytes (effector/target ratio of around2/1) are incubated for 16 h at 37° C. in the presence of variousconcentrations of polyvalent immunoglobulins (IVIg) (Tegeline®). Thecytotoxic activity induced by the antibodies is then measured bycolorimetry in quantifying in the supernatants the haemoglobin releasedby the lysed erythrocytes. The results of specific lysis are expressedin lysis percentage.

The results of FIG. 2 indicate that in the presence of macrophages, theEMABling R297 antibody has a strong remaining ADCC activity in thepresence of significant concentrations of IVIg, contrary to the AD1antibody which solely induces lysis by ADCC in the absence of IVIg.Therefore, in the absence of polyvalent immunoglobulins, the two anti-Dantibodies, EMABling R297 and AD1 have an ADCC activity of the order of29%. On the contrary, at the concentration of 5 mg/ml of polyvalentimmunoglobulins, the EMABling antibody appears at least 20 times moreactive (23% lysis versus 1% with AD1). This advantage subsists atstronger concentrations of polyvalent immunoglobulins (25 mg/ml), therespective percentages of lysis for the EMABling and AD1 antibodiesbeing 16 and 1%.

Example 5 Anti-D/Erythrocytes Rh+/Macrophages ADCC Activity. Role ofPolyvalent IVIg (Tegeline®)

According to the same protocol as described in example 4, the ADCCactivity of anti-CD20 was also studied in the presence of Raji cells andmacrophages (differentiated monocytes in M-CSF). The anti-CD20antibodies (produced by the R603 clone or Rituxan) are incubated in thepresence of macrophages, Raji cells and various concentrations ofpolyvalent IVIg (Tegeline®). After 16 h of incubation at 37° C., theADCC activity induced by the antibodies is measured by colorimetry inquantifying in the supernatants the quantity of intracellular LDH(lactate deshydrogenase) released by the Raji cells. The results ofspecific lysis are expressed in lysis percentage.

The results indicate that the anti-CD20 R603 antibody has an ADCCactivity of at least 2 times greater than that induced by Rituxan in thepresence of macrophages expressing CD16 and Tegeline®. This ADCCactivity depends from CD16 expressed by the macrophages such as shown bythe inhibitor effect of the anti-CD16 3G8.

Example 6 Anti-D/Etythrocytes Rh+/Macrophages ADCC Activity. CD16Highlighting in the Presence of IVIg

The addition of anti-CD16 antibody, 3G8, inhibits the ADCC induced bythe EMABling antibody in the presence of the strongest concentration ofIVIg tested, indicating that the induced lysis depends from the CD16expressed on the macrophages (FIG. 3).

Example 7 Phagocytosis of Rhesus+Erythrocytes by CD16+ MacrophagesInduced by the EMABling R297 Antibody in the Presence of IVIg

The capacity of anti-D R297 antibodies to induce phagocytosis ofRhesus+erythrocytes by CD16+ macrophages is studied by flow cytometry.The anti-D antibodies, the macrophages labelled with PKH67(differentiated monocytes M-CSF and Rhesus D+ erythrocytes(effector/target ratio of 5/1) labelled with PKH26 are incubated for 3 hat 4° C. and 37° C. in the presence of various concentrations ofpolyvalent IVIg (Tegeline®).

The results correspond to the percentage of PKH67/PKH26 doubly labelled,i.e. having phagocyted at least one erythrocyte.

Results: at 4° C., the macrophages and erythrocytes appear in differentwindows in cytometry, each being labelled with a specific fluorochrome.The phagocytosis percentage is very low, of the order of 4% in theabsence of IVIg, and from 1 to 2% in the presence of IVIg. These valuesat 4° C. are systematically deduced to formulate the phagocytosispercentage at 37° C.

At 37° C., the percentage of PKH67/PKH26 doubly labelled increases inthe absence of IVIg for the two assayed antibodies, R297 EMABling andAD1. In the presence of IVIg, only the EMABling antibody has thecapacity to phagocyte the Rh+erythrocytes, contrary to the AD1 antibody.Therefore, should there be 0, 1 or 2 mg/ml of IVIg, the percentage ofphagocytosis remains between 15 and 20%, showing that the addition ofIVIg does not inhibit phagocytosis induced by the EMABling antibody.

At a concentration of 1 mg/ml, the EMABling antibody is at least 5 timesgreater than the AD1 antibody. At a concentration higher than 2 mg/ml,the phagocytosis percentage is of 16.9% with the EMABling antibody andnot significant (value 0) with the AD1 antibody.

Example 8 Phagocytosis of Rhesus+Erythrocytes by CD16+ MacrophagesInduced by the R603 Antibody in the Presence of IVIg

Phagocytosis of CD20 Raji cells in the presence of CD16 macrophages,induced by the R603 antibody in the presence of IVIg was also studied.The capacity of anti-CD20 antibodies to induce phagocytosis of Rajicells by CD16+ macrophages is studied by flow cytometry. Anti-CD20antibodies, PKH67 labelled macrophages (differentiated monocytes M-CSF)and the Raji cells (effector/target ratio of 5/1, 10/1 and 20/1)labelled with PKH26 are incubated for 3 h at 4° C. and 37° C. in thepresence of various concentrations of polyvalent IVIg (Tegeline®).

The results correspond to the percentage of PKH67/PKH26 doubly labelled,having phagocyted at least one Raji cell.

Results: At 4° C., the macrophages and Raji cells appear in differentwindows in cytometry, each being labelled by a specific fluorochrome.The percentage of phagocytosis is very low, less than 5% in the absenceand in the presence of IVIg. These values at 4° C. are systematicallydeduced to formulate the phagocytosis percentage at 37° C. At 37° C.,the percentage of PKH67/PKH26 doubly labelled increases in the absenceof IVIg for the two antibodies tested, anti-CD20 R603 and Rituxan.

In the presence of IVIg, the EMABling antibody has a greater capacity,of the order of 2 times, 4 times, or even 10 times for phagocyting theRaji cells when compared to the Rituxan antibody. Therefore, shouldthere be 0; 1 or 2 mg/ml of IVIg, the percentage of phagocytosis alwaysremains greater than that induced by Rituxan, showing that in thepresence of IVIg the EMABling antibody induces phagocytosis in thepresence of CD16+ Macrophages.

1. A kit of parts for treating a malignant pathology, an auto-immunedisease or an infectious disease, comprising an effector cell whichexpresses the FcγRIII receptor (CD16) on its surface, and a monoclonalantibody, in which the affinity of the Fc region of said monoclonalantibody for CD16 is greater than the affinity of the Fc region of thepolyclonal immunoglobulins for CD16.
 2. The kit of parts according toclaim 1, wherein said effector cell which expresses the FcγRIII receptor(CD16) on its surface is a monocyte or a monocyte or monocyte precursorderived cell which expresses the FcγRIII receptor (CD16) on its surface.3. The kit of parts according to claim 1, wherein said monocyte ormonocyte precursor derived cell which expresses CD16 on its surface isselected from monocytes expressing CD16, macrophages, Natural Killercells (NK), dendritic cells and all Peripheral Blood Mononuclear Cell(or PBMC).
 4. The kit of parts according to claim 3, wherein saidmonocyte or monocyte precursor derived cell, which expresses CD16 on itssurface is a macrophage.
 5. The kit of parts according to claim 1,wherein said monoclonal antibody is not displaced by polyclonalimmunoglobulins, particularly those present in human serum, due to saidaffinity of the Fc region of said monoclonal antibody for CD16.
 6. Thekit of parts according to claim 1, wherein said monoclonal antibodybinds CD16 of said monocyte or monocyte precursor derived cell with anaffinity greater than 2.10⁶ M⁻¹.
 7. The kit of parts according to claim1, wherein said monoclonal antibody is produced in the form of amonoclonal antibodies composition, wherein each antibody has N-linkedsugar chains linked at the Fcγ glycosylation site (asparagine 297,according to Kabat), and wherein among all the N-linked sugar chains atsaid glycosylation site of all the antibodies of said composition, therate of fucose is less than 65%.
 8. The kit of parts according to claim1, wherein said monoclonal antibody is directed against an antigenselected from the 5C5 antigen (tumorous antigen expressed by the cellsof renal carcinomas), BCR (B Cell Receptor), an idiotype such as that ofanti-FVIII inhibitor antibodies, TCR (T Cell Receptor), CD2, CD3, CD4,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD45, CD30, CD33,CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66 (a, b, c, d), CD74,CD80, CD86, CD126, CD138, CD154, MUC1 (Mucine 1), MUC2 (Mucine 2), MUC3(Mucine 3), MUC4 (Mucine 4), MUC16 (Mucine 16), HM1.24 (specific antigenfor plasmocytes which is overexpressed in multiple myelomas), tenascin(protein of the extra-cellular matrix), GGT(gamma-glutamyltranspeptidase), VEGF (Vascular Endothelial GrowthFactor), EGFR (Endothelial Growth Factor receptor), CEA(carcinoembryonic antigen), CSAp (colon-specific antigen-p), ILGF(Insulin-Like Growth factor), placental growth factor, Her2/neu,carbonic anhydrase IX, IL-6, S100 proteins (multigenic family ofproteins binding to calcium), MART-1 (tumorous differentiation antigenassociated with melanoma), TRP-1 (tyrosinase-related protein 1), TRP-2(tyrosinase-related protein 2), gp100 (glycoprotein 100 kDa), amyloidproteins, rhesus D antigen, MHC molecules of class I and II such asHLA-DR), an antigen resulting from the expression of mutated genes,especially oncogenes or tumour-suppressor genes, an antigen derived fromoncogenic viruses expressed by certain well defined tumours, anubiquitous antigen overexpressed in some tumours and slightly expressedin some normal tissues such as for example the type II receptor of theMüllerian hormone, a glycosylated or non-glycosylated protein, aphospholipid, a molecule of the self or of the non-self expressed orexposed at the membrane by infected cells such as phosphatidylserine,and a protein expressed or secreted by a pathogenic agent (bacterialtoxin, proteins complexes of the bacterial or parasitic wall, viralenvelope glycoproteins, for example of HIV virus, HBV, HCV and RSV). 9.The kit of parts according to claim 8, wherein said monoclonal antibodyis directed against CD20.
 10. The kit of parts according to claim 9,wherein said anti-CD20 antibody is produced by the cell line R509deposited to the CNCM under the accession number I-3314, or by the cellline R603, deposited to the CNCM under the accession number I-3529. 11.The kit of parts according to claim 1, for use in therapy,simultaneously, sequentially or separately.
 12. The kit of partsaccording to claim 1, wherein said effector cell expressing CD16 on itssurface has a cytotoxic activity over the target cell of said antibodywhich is favoured with the interaction of the antibody with CD16. 13.The kit of parts according to claim 1, wherein said monoclonal antibodyinduces cytotoxicity by ADCC activity or by phagocytosis of said targetcell of the antibody in the presence of an effector cell expressingCD16.
 14. A pharmaceutical composition containing a kit of partsaccording to claim 1, and pharmaceutically acceptable excipients.
 15. Ause of a kit of parts according to claim 1, for manufacturing a drug.16. A use of a kit of parts according to claim 1, for manufacturing adrug for treating a malignant pathology.
 17. The use of a kit of partsaccording to claim 16, for treating a malignant pathology selected fromsolid tumours and malignant haemopathies.
 18. The use of a kit of partsaccording to claim 17, wherein the solid tumours are selected frommelanomas, carcinomas, sarcomas, gliomes and skin cancers.
 19. The useof a kit of parts according to claim 18, wherein the carcinomas areselected in the group constituted by kidney, breast, oral cavity, lungs,gastro-intestinal tract, ovaries, prostate, uterus, bladder, pancreas,liver, gallbladder, skin and testicles carcinomas.
 20. The use of a kitof parts according to claim 17, wherein the malignant haemopathies areselected from the lymphoproliferative, myeloproliferative,myelodysplasic syndromes and acute myeloid leukemias with type B NHL,acute or chronic B lymphoid leukemias, Burkitt's lymphoma,tricholeucocyte leukaemia, acute and chronic myeloid leukemias, Tlymphomas and leukemias, Hodgkin's lymphomas, Waldenström'smacroglobulinemia and multiple myelomas.
 21. A use of a kit of partsaccording to claim 1, for manufacturing a drug intended for treating anauto-immune disease and/or primitive or secondary inflammatory disease,which is specific for organs or systemic and which is associated or notwith pathogenic auto-antibodies.
 22. The use of a kit of parts accordingto claim 21, for treating an auto-immune disease and/or a primitive orsecondary inflammatory disease, which is specific for organs or systemicand which is associated or not with pathogenic auto-antibodies, selectedfrom the organ grafts rejection, the graft versus host disease,rheumatoid polyarthritis, disseminated lupus erythematosus,sclerodermia, primitive Sjögren's syndrome (or Gougerot-Sjögrensyndrome), auto-immune polyneuropathies such as multiple sclerosis, typeI diabetes, auto-immune hepatitis, ankylosing spondylarthritis, Reiter'ssyndrome, gout arthritis, coeliac disease, Crohn's disease, Hashimoto'sthyroiditis, Addison's disease, auto-immune hepatitis, Basedow'sdisease, ulcerative colitis, vasculitis such as systemic vasculitisassociated with ANCA (Antineutrophil cytoplasmic antibody), auto-immunecytopenias and other haematological complications in adults andchildren, such as acute or chronic auto-immune thrombopenias,auto-immune haemolytic anaemias, haemolytic disease of the newborn(HDN), cold agglutinin disease, thrombocytopenic thrombotic purpura andacquired auto-immune haemophilia; Goodpasture's syndrome,extra-membraneous nephropathies, auto-immune bullous skin disorders,refractory myasthenia, mixed cryoglobulinemias, psoriasis, juvenilechronic arthritis, inflammatory myositis, dermatomyositis andauto-immune systemic diseases in children including antiphospholipidssyndrome.
 23. A use of a kit of parts according to claim 1, formanufacturing a drug for treating an infectious disease.
 24. The use ofa kit of parts according to claim 23, for treating an infectious diseaseselected from those induced by virus (human immunodeficiency virus orHIV, virus of hepatitis B or C (HBV, HCV), Epstein-Barr virus or EBV,cytomegalovirus or CMV, enterovirus, influenza with Influenza virus A, Band C, syncytial respiratory virus or SRV, or HTLV), bacteria and/ortheir toxins (tetanus, diphtheria, pneumococci, meningococci,staphylococci including methicilin resistant forms, Klebsiellas,Shigellas, pseudomonas aeruginosa, enterobacteria or antibioticresistant pathologies including nosocomial diseases), parasites(paludism, leishmaniosis, trypanosomiasis) as well as emerging diseases,for example Chikungunya, bird flu, severe acute respiratory virussyndrome or SARS, virus responsible for haemorrhagic fevers such asEbola or Dengue fever or west Nile virus, and those related tobio-terrorism, such as Anthrax, Botulism, Plague, smallpox and poxvirus,Tularaemia, haemorrhagic fever agents, brucellosis, B Enterotoxins ofStaphylococcus, diphtheric toxin or viral Encephalitis.